AMPD2 Antibody

What is AMPD2 and what is its biological significance?

AMPD2 (Adenosine Monophosphate Deaminase 2) is an enzyme that catalyzes the deamination of AMP to IMP and plays a critical role in the purine nucleotide cycle. It is essential for cellular energy homeostasis and nucleic acid synthesis. AMPD2 is mainly expressed in non-muscle tissues, with particularly high expression in the liver and cerebellum. It plays a crucial role in neurogenesis and neuronal differentiation, with mutations linked to neurodegenerative disorders, particularly pontocerebellar hypoplasia type 9 (PCH-9) . Recent research has also identified AMPD2 as being overexpressed in colorectal cancer, suggesting a potential role in cancer metabolism .

What applications are AMPD2 antibodies typically used for?

AMPD2 antibodies are primarily used for:

• Western Blot (WB): For detection of denatured AMPD2 protein (typical dilutions range from 1:500-1:2400)

• Immunohistochemistry (IHC): For visualization in tissue sections (typical dilutions range from 1:100-1:400)

• Immunofluorescence/Immunocytochemistry (IF/ICC): For cellular localization studies

• ELISA: For quantitative analysis

The antibody selection should be based on the specific application and sample type to be analyzed.

What are the common tissue samples used for AMPD2 antibody validation?

Based on published validation data, the following tissues and cells have been successfully used:

• Human liver tissue: Shows strong AMPD2 expression in Western blot applications

• Human cerebellum tissue: Recommended for IHC applications

• HeLa cell lysates: Used for Western blot validation

• N87 xenografts: Used for IHC validation

• Leukocytes: Can be used for enzymatic activity assays

For researchers planning initial experiments, these tissues represent validated starting points with confirmed AMPD2 expression.

How should I optimize Western blot protocols for AMPD2 detection?

For optimal Western blot results with AMPD2 antibodies:

For challenging samples, consider longer primary antibody incubation (overnight at 4°C) and optimization of antigen retrieval methods if signal is weak .

What controls should be included when using AMPD2 antibodies?

To ensure experimental validity, include the following controls:

• Positive tissue controls: Human liver tissue or cerebellum tissue samples known to express AMPD2

• Negative controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG)

  • Tissues known to have low AMPD2 expression

• Loading controls: Use housekeeping proteins (β-actin, GAPDH) for Western blots

• Knockdown/knockout validation: When possible, include AMPD2 siRNA-treated samples as specificity controls

• Blocking peptide controls: For polyclonal antibodies, pre-incubation with the immunizing peptide should abolish specific signal

These controls help distinguish specific from non-specific signals and validate antibody performance across experiments.

How can I assess AMPD2 enzyme activity rather than just protein expression?

Beyond immunodetection, functional assays for AMPD2 activity include:

• Spectrophotometric enzyme assay: Measure the conversion of AMP to IMP by monitoring changes in absorbance at 340 nm

• Nucleotide profiling: Quantify cellular adenosine and guanine nucleotide levels using HPLC or LC-MS/MS

• Adenosine challenge assay: Treating cells with adenosine (10-50 μM) and measuring ATP/GTP ratios can reveal AMPD2 functional status

• Functional rescue experiments: Adding guanosine to cells with AMPD2 deficiency can rescue cellular phenotypes if the defect is due to GTP depletion

For accurate enzymatic activity measurements, fresh samples are essential as enzyme activity can degrade during storage .

How do mutations in AMPD2 affect cellular functions, and how can I study these effects?

AMPD2 mutations, particularly those associated with PCH-9, lead to several measurable cellular phenotypes that can be studied:

• Adenosine-induced toxicity: AMPD2-deficient cells show increased sensitivity to adenosine treatment (10-50 μM), which can be quantified through viability assays

• Nucleotide imbalance:

  • Elevated ATP levels (quantifiable by HPLC or luminescence assays)

  • Reduced GTP levels (by ~50% in patient-derived cells)

• Protein synthesis defects:

  • Reduced global protein synthesis rates (measurable by puromycin incorporation assays)

  • Impaired GTP-dependent translation initiation (analyze polysome profiles)

• Rescue experiments:

  • Treatment with 5-Iodotubercidin (adenosine uptake inhibitor) can block adenosine toxicity

  • Supplementation with guanosine can restore guanine nucleotide pools and rescue cellular phenotypes

These assays enable mechanistic studies of AMPD2 function in normal and disease states.

How can I use AMPD2 antibodies to study its role in neurodegeneration?

For neurodegenerative disease research, consider these specialized approaches:

• Patient-derived iPSC models:

  • Generate induced pluripotent stem cells from patients with AMPD2 mutations

  • Differentiate into neural progenitor cells (NPCs) and neurons

  • Use AMPD2 antibodies to assess expression levels and localization in these models

• Brain section IHC analysis:

  • Use AMPD2 antibodies on brain sections (particularly cerebellum) with antigen retrieval using TE buffer pH 9.0

  • Compare expression patterns between normal and PCH-9 patient samples

  • Perform co-localization studies with neuronal markers

• Adenosine sensitivity assays:

  • Neural cells with AMPD2 deficiency show increased vulnerability to adenosine

  • Monitor cell growth restriction and apoptosis markers after adenosine treatment

• Developmental analysis:

  • Track AMPD2 expression during neuronal differentiation using IF/ICC

  • Correlate expression levels with developmental milestones and neurogenesis markers

These approaches can reveal how AMPD2 dysfunction contributes to neurodegeneration mechanisms.

What is the relationship between AMPD2 and the Notch signaling pathway in cancer progression?

Recent research has identified connections between AMPD2 and Notch signaling in colorectal cancer:

• Co-expression analysis:

  • Use dual IHC or IF with AMPD2 and Notch3 antibodies (1:200 dilution) to analyze co-expression patterns in cancer tissues

  • Quantify correlation between AMPD2 and Notch pathway components using image analysis software

• Mechanistic studies:

  • After AMPD2 knockdown or overexpression, measure changes in Notch pathway components (Notch3, HES1, HEY1) by Western blot or qPCR

  • Determine if AMPD2 effects on cancer cell proliferation can be rescued or blocked by Notch pathway modulators

• Transcriptomic analysis:

  • Analyze gene expression patterns in AMPD2-high versus AMPD2-low tumors (as shown in TCGA data)

  • Perform pathway enrichment analysis to identify signaling networks affected by AMPD2 expression

This research direction may reveal novel therapeutic targets for cancers with AMPD2 overexpression.

Why might I observe different molecular weights for AMPD2 in Western blot experiments?

While the calculated molecular weight of AMPD2 is 101 kDa, researchers may observe bands at different positions:

• Multiple isoforms: AMPD2 has multiple isoforms with molecular masses ranging from 88-101 kDa

  • Isoform L: 879 aa, 101 kDa

  • Other isoforms: ~798 aa, 92 kDa

• Post-translational modifications: Phosphorylation or other modifications may alter migration patterns

• Proteolytic processing: Sample preparation conditions may affect protein integrity

• Antibody specificity: Different antibodies may recognize specific epitopes present in certain isoforms but not others

To address variability:

• Use positive control samples with confirmed AMPD2 expression

• Compare results across multiple antibodies targeting different epitopes

• Consider tissue-specific expression patterns of different isoforms

How can I optimize IHC protocols for AMPD2 detection in different tissue types?

For optimal IHC results with AMPD2 antibodies across different tissues:

Tissue-specific considerations:

• Cerebellum: Requires careful antigen retrieval optimization due to dense tissue architecture

• Liver: Generally shows strong signal with standard protocols

• Cancer tissues: May show heterogeneous expression requiring careful analysis

What approaches can resolve contradictory results when using different AMPD2 antibodies?

When facing inconsistent results with different AMPD2 antibodies:

• Epitope mapping analysis:

  • Compare the immunogens/epitopes targeted by each antibody

  • Antibodies targeting different regions (N-terminal vs. internal vs. C-terminal) may give different results

  • Chart the epitope locations relative to functional domains and isoform variations

• Multi-technique validation:

  • Confirm results using orthogonal methods (e.g., if WB and IHC give different results, add IF or ELISA)

  • Use genetic approaches (siRNA knockdown, CRISPR knockout) to validate specificity

• Sample-specific considerations:

  • Different fixation methods may affect epitope availability

  • Certain tissues may express specific isoforms recognized by only some antibodies

  • Post-translational modifications may mask epitopes in a context-dependent manner

• Functional correlation:

  • Correlate antibody staining patterns with known AMPD2 activity measurements

  • Use enzymatic assays to confirm protein detection results

This systematic approach can reconcile seemingly contradictory results and provide more reliable data interpretation.

How can AMPD2 antibodies be used to explore therapeutic approaches for PCH-9?

For investigating potential PCH-9 treatments using AMPD2 antibodies:

• Therapeutic screening platforms:

  • Use AMPD2 antibodies to monitor protein levels/localization in patient-derived cells treated with candidate compounds

  • Track changes in downstream signaling pathways affected by AMPD2 dysfunction

• Biomarker development:

  • Establish correlations between AMPD2 protein levels/localization and disease severity

  • Monitor treatment efficacy using AMPD2 as a biomarker

• Rescue experiments:

  • Use AMPD2 antibodies to validate the efficacy of nucleoside supplementation therapy (particularly guanosine)

  • Monitor restoration of normal protein synthesis and neuronal survival

• Gene therapy validation:

  • Use AMPD2 antibodies to confirm successful gene replacement/correction approaches

  • Quantify restoration of normal AMPD2 expression levels and localization patterns

These approaches could accelerate development of treatments for this currently incurable neurological disorder.

What are the technical considerations when studying AMPD2 in the context of cancer metabolism?

When investigating AMPD2's role in cancer metabolism:

• Metabolic profiling integration:

  • Combine AMPD2 protein expression analysis with metabolomic profiling of purine metabolites

  • Correlate AMPD2 levels (by WB/IHC) with adenosine/guanosine ratios and energy charge measurements

• Tumor microenvironment considerations:

  • Use multiplex IHC to simultaneously visualize AMPD2 expression and metabolic markers

  • Assess AMPD2 expression in hypoxic vs. normoxic tumor regions

• In vivo models:

  • Use AMPD2 antibodies to monitor expression in xenograft models

  • Track changes in AMPD2 levels during tumor progression and in response to therapy

• TCGA data correlation:

  • Use antibody validation data to interpret transcriptomic data from cancer databases

  • Develop AMPD2-based signatures that correlate with patient outcomes

These approaches can provide insights into AMPD2's role in cancer metabolism and identify potential therapeutic vulnerabilities.

How can advanced imaging techniques enhance AMPD2 localization studies?

Leverage cutting-edge imaging approaches for detailed AMPD2 localization studies:

• Super-resolution microscopy:

  • Use AMPD2 antibodies with compatible fluorophores for STED, PALM, or STORM microscopy

  • Achieve nanoscale resolution of AMPD2 distribution within subcellular compartments

• Live-cell imaging:

  • Develop cell-permeable AMPD2 antibody fragments or nanobodies

  • Track dynamic changes in AMPD2 localization during cellular responses

• Proximity labeling approaches:

  • Combine AMPD2 antibodies with proximity ligation assays (PLA)

  • Identify protein-protein interactions involving AMPD2 in situ

• Correlative light-electron microscopy (CLEM):

  • Use AMPD2 antibodies compatible with both fluorescence and electron microscopy

  • Achieve high-resolution ultrastructural localization of AMPD2

• Spatial transcriptomics integration:

  • Correlate AMPD2 protein expression with spatial gene expression patterns

  • Identify microenvironmental factors influencing AMPD2 expression

These advanced techniques can reveal previously undetectable aspects of AMPD2 biology in normal and pathological contexts.